Age-related alterations to NADPH oxidase and glial cell activation after spinal cord injury

 描述(申请人提供)：美国目前有超过25万人患有与脊髓损伤(SCI)相关的残疾。自20世纪80年代S以来，老年人口中脊髓损伤的数量一直在稳步增加，并与较高的并存率有关。氧化应激和活性氧物种(ROS)在老化的组织中增加，并与中枢神经系统(CNS)的组织损伤和慢性炎症有关。这些应激源影响神经胶质细胞的活动，导致老年人群脊髓损伤恢复延迟。到目前为止，还没有研究过与年龄相关的神经胶质细胞对脊髓损伤反应的变化。NADPH氧化酶(NOX)家族被认为通过产生ROS在小胶质/巨噬细胞活化、炎症和组织损伤中发挥调节作用，并在脊髓损伤后慢性上调。在人类和啮齿动物老年人群中，NOx、小胶质细胞和星形胶质细胞都显示出改变的轮廓，炎性形态和基本激活状态增加。因此，我们假设与年龄相关的NOX活性和表达上调导致促炎神经胶质细胞激活状态增加，从而导致过度的神经胶质反应和脊髓损伤的恢复。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将表征衰老大鼠脊髓中基本的胶质细胞激活状态以及NOX的表达和活性。为此，我们将利用免疫组织化学和生物化学双标技术对3月龄和12月龄大鼠的NOX和胶质细胞激活状态进行研究。在目标2中，我们将展示衰老对脊髓鳕鱼损伤后NOX表达和活性、神经胶质细胞激活、损伤大小和功能恢复的影响。为此，我们将评估脊髓中度挫伤后NOX和神经胶质细胞的激活。 3月龄和12月龄大鼠进行免疫组织化学、生化、RT-qPCR和功能评定。在目标3中，我们将评估抑制NOX活性和表达对基础胶质细胞激活和损伤反应的影响。为此，我们将使用一种NOX2特异性抑制剂来评估NOX对老年大鼠小胶质细胞和星形胶质细胞激活的调节作用。这种作用将通过免疫组织化学、生物化学、亚细胞分级和功能评估来研究。这些目标产生的数据将有助于指导研究人员和临床医生进行更有效的诊断，并可能为老龄化人口中脊髓损伤未来的潜在治疗靶点提供重要的见解。

 DESCRIPTION (provided by applicant): Over 250,000 people in the US are currently living with disabilities related to spinal cord injury (SCI). The number of SCI's among the aging population has been steadily increasing since the 1980's, and is associated with high rates of co-morbidities. Oxidative stress and reactive oxygen species (ROS) are increased in aging tissue and have been causally implicated in tissue damage and chronic inflammation in the central nervous system (CNS). These stressors influence the activity of glial cells, contributing to the delayed recovery to SCI seen in the aging population. No work to date has examined age-related alterations in glial cell response to SCI. The NADPH oxidase (NOX) family of enzymes is suggested to play a modulatory role in microglial/macrophage activation, inflammation and tissue damage through the production of ROS, and is chronically up-regulated after SCI. NOX, microglia and astrocytes all show altered profiles with increased inflammatory morphology and basal activation states in both human and rodent aged populations. Thus, we hypothesize that age-related upregulation in NOX activity and expression leads to increased proinflammatory glial cell activation states, resulting in exaggerated glial responses and diminished recovery to spinal cord injury. To test this hypothesis, we propose three specific aims. In aim 1, we will characterize basal glial cell activation states and NOX expression and activity in the aging rodent spinal cord. In this aim, we will use double labeled immunohistochemistry and biochemical techniques to characterize NOX and glial cell activation state in 3 and 12 month aged rats. In aim 2, we will demonstrate the effects of aging on NOX expression and activity, glial cell activation, lesion size and functional recovery after spinal cod injury. In this aim, we will assess NOX and glial cell activation after a moderate contusion SCI in 3 and 12 month aged rats using immunohistochemistry, biochemistry, RT-qPCR and functional assessment. In aim 3, we will evaluate the effect of inhibition of NOX activity and expression on basal glial activation and response to injury. In this aim, we will assess the modulatory effects o NOX on microglia and astrocyte activation in aged rats using a NOX2 specific inhibitor. This effect will be investigated using immunohistochemistry, biochemistry, subcellular fractionation and functional assessment. The data generated in these aims will help to guide researchers and clinicians in more effective diagnosis and may provide important insight into potential future therapeutic targets for SCI in the aging population.